Butyrylcholinesterases having an enhanced ability to hydrolyze acyl ghrelin

ABSTRACT

This document provides butyrylcholinesterases having an enhanced ability to hydrolyze acyl ghrelin as well as nucleic acids encoding such butyrylcholinesterases. This document also provides methods and materials for treating obesity and/or aggression. For example, methods for administering a nucleic acid encoding a wild-type or mutant butyrylcholinesterase having the ability to hydrolyze acyl ghrelin to a mammal under conditions wherein the level of acyl ghrelin within the mammal is reduced, under conditions wherein the rate of body weight gain of the mammal is reduced, under conditions wherein the mammal&#39;s level of aggression is reduced, and/or under conditions wherein the mammal&#39;s rate of developing stress-induced tissue damage are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.15/307,665, filed Oct. 28, 2016 (now U.S. Pat. No. 10,301,609), which isa National Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2015/028141, having an International Filing Dateof Apr. 29, 2015, which claims the benefit of U.S. Provisional Ser. No.61/985,883 filed Apr. 29, 2014. This disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

BACKGROUND 1. Technical Field

This document relates to butyrylcholinesterases (BChE) having anenhanced ability to hydrolyze acyl ghrelin as well as methods andmaterials for treating obesity and aggression. For example, thisdocument provides nucleic acids encoding a butyrylcholinesterase havingan enhanced ability to hydrolyze acyl ghrelin. In addition, thisdocument provides methods and materials for using vectors (e.g., viralvectors) to express butyrylcholinesterases under conditions that reducebody weight within a mammal, reduce body weight gain in a mammal, reducea mammal's level of aggression, reduce a mammal's response to cocaine,and/or reduce the severity of a stress-induced disability.

2. Background Information

Obesity is a medical condition where excess body fat accumulated to theextent that can cause a negative effect on health and/or lead to reducedlife expectancy.

An aggressive behavior can be a behavior that is characterized by strongself-assertion with hostile or harmful tones. Aggressive behaviors canlead to various problems such as academic, employment, and relationshipproblems.

Stress-related disability is a growing problem in advanced countrieswith older populations.

SUMMARY

This document provides butyrylcholinesterases having an enhanced abilityto hydrolyze acyl ghrelin. For example, this document provides nucleicacids that encode a butyrylcholinesterase polypeptide that (a) containsone or more amino acid mutations with respect to a wild-typebutyrylcholinesterase polypeptide and (b) exhibits an elevated abilityto hydrolyze acyl ghrelin with respect to the ability of that wild-typebutyrylcholinesterase polypeptide. This document also provides methodsand materials for treating obesity and/or aggression. For example, thisdocument provides methods for administering a nucleic acid encoding awild-type or mutant butyrylcholinesterase having the ability tohydrolyze acyl ghrelin to a mammal under conditions wherein the level ofacyl ghrelin within the mammal is reduced, under conditions wherein thebody weight of the mammal is reduced, under conditions wherein the bodyweight gain of the mammal is reduced, under conditions wherein themammal's level of aggression is reduced, under conditions wherein themammal's response to cocaine is reduced, and/or under conditions whereinthe severity of a stress-induced disability is reduced. This documentalso provides methods for reducing stress-induced reactions by, forexample, reducing the numbers of cells expressing “senescence-relatedmarkers” such as beta galactosidase.

As described herein, a nucleic acid can be designed to encode apolypeptide that includes the amino acid sequence set forth in FIG. 1 orthe amino acid sequence set forth in FIG. 1 with one or more (e.g., two,three, four, five, or six) of the following amino acid substitutions:A199S, S227A, S287G, A328W, F329M, or Y332G (amino acid numbering startsafter the signal sequence). In some cases, such a polypeptide can havean increased ability to hydrolyze acyl ghrelin as compared to apolypeptide having the amino acid sequence set forth in FIG. 1. In somecases, a nucleic acid can be designed to encode a polypeptide thatincludes the amino acid sequence set forth in FIG. 2 or the amino acidsequence set forth in FIG. 2 with one or more (e.g., two, three, four,five, or six) of the following amino acid substitutions: A199S, F227A,S287G, A328W, F329M, or Y332G (amino acid numbering starts after thesignal sequence). In some cases, such a polypeptide can have anincreased ability to hydrolyze acyl ghrelin as compared to a polypeptidehaving the amino acid sequence set forth in FIG. 2.

As also described herein, a wild-type or mutant butyrylcholinesterase ornucleic acid encoding a wild-type or mutant butyrylcholinesterase can beadministered to a mammal (e.g., an obese human or an aggressive human)to reduce or control the body weight gain of that mammal, especiallywhen, for example, that mammal has ready access to rich food (e.g., highcalorie or high fat food), to reduce the aggressiveness of that mammal,and/or to reduce stress induced biochemical changes in body tissue. Forexample, a viral vector encoding a mutant butyrylcholinesterase havingan enhanced ability to hydrolyze acyl ghrelin as compared to a wild-typehuman butyrylcholinesterase can be administered to a human to reduce thebody weight of that human or to reduce the aggressiveness of that humanor to protect that human from stress-related damage. In some cases,expression of a wild type or mutant BChE in vivo can reduce external andinternal signs of aging and lower stress-induced tissue damage.

In general, one aspect of this document features a nucleic acid encodinga polypeptide having the amino acid sequence set forth in SEQ ID NO:3with a F329M substitution (amino acid numbering starts after the signalsequence) or with a combination with one or more of the following aminoacid substitutions: A199S, F227A, S287G, A328W, F329M, or Y332G (aminoacid numbering starts after the signal sequence), wherein thepolypeptide has an increased ability to hydrolyze acyl ghrelin ascompared to a polypeptide having the amino acid sequence set forth inSEQ ID NO:3.

In another aspect, this document features a viral vector comprising anucleic acid sequence that encodes a polypeptide having the amino acidsequence set forth in SEQ ID NO:3 with a F329M substitution (amino acidnumbering starts after the signal sequence) or with a combination withone or more of the following amino acid substitutions: A199S, F227A,S287G, A328W, F329M, or Y332G (amino acid numbering starts after thesignal sequence), wherein the polypeptide has an increased ability tohydrolyze acyl ghrelin as compared to a polypeptide having the aminoacid sequence set forth in SEQ ID NO:3.

In another aspect, this document features a polypeptide having the aminoacid sequence set forth in SEQ ID NO:3 with a F329M substitution (aminoacid numbering starts after the signal sequence) or with a combinationwith one or more of the following amino acid substitutions: A1995,F227A, S287G, A328W, F329M, or Y332G (amino acid numbering starts afterthe signal sequence), wherein the polypeptide has an increased abilityto hydrolyze acyl ghrelin as compared to a polypeptide having the aminoacid sequence set forth in SEQ ID NO:3.

In another aspect, this document features a method for reducing the bodyweight of a mammal (e.g., a mammal having wild-typebutyrylcholinesterase). The method comprises, or consists essentiallyof, administering a polypeptide or a nucleic acid encoding thepolypeptide to the mammal, wherein the polypeptide comprises the abilityto hydrolyze acyl ghrelin, and wherein the body weight of the mammal isreduced following the administration. The mammal can be a human. Themethod can comprise administering the polypeptide to the mammal. Themethod can comprise administering the nucleic acid to the mammal. Themethod can comprise administering a viral vector comprising the nucleicacid to the mammal. The viral vector can be an adeno-associated virusvector.

In another aspect, this document features a method for reducing theaggressiveness of a mammal (e.g., a mammal having wild-typebutyrylcholinesterase). The method comprises, or consists essentiallyof, administering a polypeptide or a nucleic acid encoding thepolypeptide to the mammal, wherein the polypeptide comprises the abilityto hydrolyze acyl ghrelin, and wherein the aggressiveness of the mammalis reduced following the administration. The mammal can be a human. Themethod can comprise administering the polypeptide to the mammal. Themethod can comprise administering the nucleic acid to the mammal. Themethod can comprise administering a viral vector comprising the nucleicacid to the mammal. The viral vector can be an adeno-associated virusvector.

In another aspect, this document features a method for reducing the rateof aging in terms of external appearance and internal development ofstress-induced tissue damage and biochemical and cellular changescharacteristic of senescence in a mammal (e.g., a mammal havingwild-type butyrylcholinesterase). The method comprises, or consistsessentially of, administering a polypeptide or a nucleic acid encodingthe polypeptide to the mammal, wherein the polypeptide comprises theability to hydrolyze acyl ghrelin.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a sequence listing of a wild-type mouse butyrylcholinesterase(SEQ ID NO:1) along with a nucleic acid sequence (SEQ ID NO:2) thatencodes this wild-type mouse butyrylcholinesterase.

FIG. 2 is a sequence listing of a wild-type human butyrylcholinesterase(SEQ ID NO:3) along with a nucleic acid sequence (SEQ ID NO:4) thatencodes this wild-type human butyrylcholinesterase.

FIGS. 3A-3D. Deacylation of human ghrelin by human BChE. (A) One μg ofpurified human BChE was analyzed by SDS-PAGE and stained with SYPRO-Rubyto confirm high purity (single major band). (B) One ng of humanacyl-ghrelin was treated with different amount of human BChE. After 20hours of incubation, residual acyl ghrelin and desacyl ghrelin in eachreaction were determined. (C) One ng of human acyl ghrelin was treatedwith 10 μg of human BChE and % decrease in residual acyl ghrelin wasmeasured as a function of time. (D) Ten μg of human BChE was incubatedfor 10 minutes with 0, 10, or 100 μM of BChE inhibitor, iso-OMPA, or aproteinase inhibitor mixture containing 1 μM of aprotinin, 20 μM ofleupeptin, and 15 mM of pepstatin A. Afterwards, 1 ng of human acylghrelin was added, and the reaction was incubated for 20 hours. Thehydrolysis activities with butyrylthiocholine (BTCh) or acyl-ghrelin assubstrates were determined by Ellman assay and acyl-ghrelin immunoassay,respectively. Data are normalized to the no inhibitor controls. Allvalues are means ±SD, each with duplicate samples (n=3).

FIG. 4. Circulating levels of acyl-ghrelin and desacyl-ghrelin in micewith gene transfer of native mouse BChE (mBChE wt) or mutated enzyme(mBChE mut). C57b1/6 mice at 12-week age were fasted for 20 hours, andserum samples were collected to determine the levels of acyl ghrelin anddesacyl ghrelin. Samples from ad libitum fed mice served as controls.All values are means ±SD (n=5 per group), ***, p<0.001 compared withother groups.

FIG. 5. Faster removal of ghrelin injected into mice with BChE genetransfer compared with a control gene transfer. Circulating acyl ghrelinlevels after injection of 1 μg of recombinant human acyl ghrelin.Control 16-week old C57b1/6 mice and mice with gene transfer of AAV-Luc(control) or AAV-mBChE mut vector treatments were used. Acyl ghrelinlevels in serum were determined 1, 3, and 10 minutes after injection.Serum samples from ad libitum fed mice served as basal levels. Allvalues are means ±SD (n=9).

FIGS. 6A-6B. Selective inhibition of BChE increases acyl ghrelin levelsin mice with AAV mBChE mut vector treatment. C57b1/6 mice with AAV-Lucor AAV-mBChE mut vector treatments (18-week old) received 40 mg kg⁻¹ ofselective BChE inhibitor (iso-OMPA) or saline. All mice were then fastedfor 6 hours, and serum samples were collected to determine (A) BChEactivity versus the reference substrate (BTCh) and (B) the levels ofacyl ghrelin. All values are means ±SD, each with duplicate samples(n=4), ***, p<0.001 compared with saline group.

FIG. 7. Mouse models with high-expression levels of BChE by genetransfer. C57b1/6 mice were injected with AAV-Luc vector, AAV-mBChE wildtype, or AAV-mBChE mutant vector at the dose of 1×10¹³ viral particlesper mouse at 6-week age. Plasma samples were collected at the indicatedtime points and assayed for BTCh hydrolysis activity. All values aremeans ±SD, each with triplicate samples (n=5 per group).

FIGS. 8A-8B are graphs plotting the weight gain of the indicated mice onnormal diet and high fat (obesogenic) diet.

FIG. 9 is a graph plotting the number of fights per testing trial forthe indicated mice.

FIG. 10 is a graph plotting the number of fights per testing trial forthe indicated mice.

FIG. 11 is a graph plotting the number of fights per testing trial forthe indicated mice.

FIG. 12 is a graph plotting the number of fights per testing trial forthe indicated mice.

FIGS. 13A-13D. Photomicrographs of fat cells in 22-month-old mice. A)Fad pad sample from untreated control mouse. Bluestain=beta-galactosidase activity, classic sign of cellular aging andsenescence. B) Sample from same-age mouse given adenoviral vector formutant mouse butyrylcholinesterase at age 1 month. Absence of blue stainindicates healthy cells. C) photograph showing the external appearanceof 16 month old mice from indicated groups (c=control, v=vector). D)Survival curve showing early death of controls housed for 5 months underconditions of moderate stress.

DETAILED DESCRIPTION

This document provides butyrylcholinesterases having an enhanced abilityto hydrolyze acyl ghrelin, nucleic acids encoding suchbutyrylcholinesterases, vectors (e.g., viral vectors) that containnucleic acid encoding such butyrylcholinesterases, and methods andmaterials for treating obesity, aggression, or both. For example, thisdocument provides nucleic acids encoding a butyrylcholinesterase havingan enhanced ability to hydrolyze acyl ghrelin. In addition, thisdocument provides methods and materials for using vectors (e.g., viralvectors) to express butyrylcholinesterases under conditions that reducebody weight gain within a mammal, that reduce a mammal's level ofaggression, and/or that reduce stress that can lead to premature agingexternally and internally, as well as increased risk of premature death.This document also provides methods and materials for using vectors toexpress wild type butyrylcholinesterases in greater than normal amountsunder conditions that reduce body weight gain within a mammal, thatreduce a mammal's level of aggression, and/or that reduce stress-inducedtissue damage.

The polypeptides provided herein can be designed to include the aminoacid sequence set forth in SEQ ID NO:1 or 3 or the amino acid sequenceset forth in SEQ ID NO:1 or 3 with the exception that it contains one,two, three, four, five, or more amino acid additions, subtractions, orsubstitutions. For example, a polypeptide provided herein can have theamino acid sequence set forth in SEQ ID NO:3 with the following sixchanges: A1995, F227A, S287G, A328W, F329M, and Y332G In some cases, apolypeptide provided herein can have the amino acid sequence set forthin SEQ ID NO:3 with a single F329M change. Other examples ofpolypeptides provided herein are set forth in Table 1. In some cases, apolypeptide provided herein can have an enhanced ability to hydrolyzeacyl ghrelin as compared to a wild type human BChE having the amino acidsequence set forth in SEQ ID NO:3.

TABLE 1 Polypeptides based on human BChE. Mutations with respect to SEQID NO: 3 A199S, F227A, S287G, and A328W F329M A199S, F227A, S287G,A328W, and F329M

In some cases, a polypeptide provided herein can have an amino acidsequence with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to areference sequence (e.g., SEQ ID NO:1 or 3). In some cases, apolypeptide provided herein can have an amino acid sequence with atleast 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:1 or 3,provided that the amino acid sequence is not identical to the sequenceset forth in SEQ ID NO:1 and 3. Percent sequence identity is calculatedby determining the number of matched positions in aligned amino acidsequences (target amino acid sequence aligned to an identified aminoacid sequence), dividing the number of matched positions by the numberof amino acids of the identified amino acid sequence (e.g., SEQ IDNO:3), and multiplying by 100. A matched position refers to a positionin which identical amino acids occur at the same position in alignedamino acid sequences. Percent sequence identity also can be determinedfor any nucleic acid sequence.

Percent sequence identity is determined by comparing a target amino acidsequence to the identified amino acid sequence (e.g., SEQ ID NO:3) usingthe BLAST 2 Sequences (B12seq) program from the stand-alone version ofBLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. Thisstand-alone version of BLASTZ can be obtained on the World Wide Web fromFish & Richardson's web site (fr.com/blast) or the U.S. government'sNational Center for Biotechnology Information web site(ncbi.nlm.nih.gov). Instructions explaining how to use the B12seqprogram can be found in the readme file accompanying BLASTZ.

B12seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: −iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); −j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); −p is set toblastn; −o is set to any desired file name (e.g., C:\output.txt); −q isset to −1; −r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\B12seq c:\seq1.txt −jc:\seq2.txt −p blastn −o c:\output.txt −q −1 −r 2. If the targetsequence shares homology with any portion of the identified sequence,then the designated output file will present those regions of homologyas aligned sequences. If the target sequence does not share homologywith any portion of the identified sequence, then the designated outputfile will not present aligned sequences.

For example, if (1) a target sequence is compared to the sequence setforth in a reference sequence that has 100 amino acid residues and (2)the B12seq program presents the target sequence aligned with a region ofthat sequence with the number of matches being 86, then the amino acidtarget sequence has a percent identity to that reference sequence thatis 86 (i.e., 86±100×100=86.0). It is noted that the percent identityvalue is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13,and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18,and 78.19 are rounded up to 78.2. It also is noted that the length valuewill always be an integer.

A polypeptide provided herein can be produced using any suitable method,including recombinant technology. In some cases, a polypeptide providedherein can be a substantially pure polypeptide. As used herein, the term“substantially pure” with reference to a polypeptide means that thepolypeptide is substantially free of other polypeptides, lipids,carbohydrates, and nucleic acid. In some cases, a substantially purepolypeptide can be a polypeptide that is at least 60 percent pure or isany chemically synthesized polypeptide. A substantially pure polypeptidecan be at least about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percentpure. Typically, a substantially pure polypeptide will yield a singlemajor band on a non-reducing polyacrylamide gel.

In some cases, a polypeptide provided herein can be modified by linkageto a polymer such as polyethylene glycol (PEG), or by fusion to anotherpolypeptide such as albumin, for example. For example, one or more PEGmoieties can be conjugated to a polypeptide provided herein via lysineresidues. Linkage to PEG or another suitable polymer, or fusion toalbumin or another suitable polypeptide can result in a modifiedpolypeptide having an increased half life as compared to an unmodifiedpolypeptide. Without being bound by a particular mechanism, an increasedserum half life can result from reduced proteolytic degradation, immunerecognition, or cell scavanging of the modified polypeptide. Anyappropriate method can be used to modify a polypeptide provided hereinby linkage to PEG (also referred to as “PEGylation”) or other polymersincluding, without limitation, those described elsewhere (U.S. Pat. No.6,884,780; Cataliotti et al., Trends Cardiovasc. Med., 17:10-14 (2007);Veronese and Mero, BioDrugs, 22:315-329 (2008); Miller et al.,Bioconjugate Chem., 17:267-274 (2006); and Veronese and Pasut, DrugDiscov. Today, 10:1451-1458 (2005). Examples of methods for modifying apolypeptide provided herein by fusion to albumin include, withoutlimitation, those described elsewhere (U.S. Patent Publication No.20040086976, and Wang et al., Pharm. Res., 21:2105-2111 (2004)).

Nucleic Acids, Vectors, and Host Cells

This document also provides nucleic acids encoding a polypeptideprovided herein as well as expression vectors containing the nucleicacids, and host cells containing the nucleic acids and/or expressionvectors. As used herein, the term “nucleic acid” refers to both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. A nucleic acid molecule can be double-stranded orsingle-stranded (i.e., a sense or an antisense single strand). Nucleicacids include, for example, cDNAs encoding the chimeric polypeptidesprovided herein.

An “isolated nucleic acid” is a nucleic acid that is separated fromother nucleic acid molecules that are present in a vertebrate genome,including nucleic acids that normally flank one or both sides of thenucleic acid in a vertebrate genome. The term “isolated” as used hereinwith respect to nucleic acids also includes any non-naturally-occurringnucleic acid sequence, since such non-naturally-occurring sequences arenot found in nature and do not have immediately contiguous sequences ina naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not considered an isolated nucleic acid.

Isolated nucleic acid molecules can be produced using standardtechniques, including, without limitation, common molecular cloning andchemical nucleic acid synthesis techniques. For example, polymerasechain reaction (PCR) techniques can be used to obtain an isolatednucleic acid containing a nucleotide sequence that encodes a BChEpolypeptide. PCR refers to a procedure or technique in which targetnucleic acids are enzymatically amplified. Sequence information from theends of the region of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified. PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. Primers typically are 14 to 40nucleotides in length, but can range from 10 nucleotides to hundreds ofnucleotides in length. General PCR techniques are described, for examplein PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler,Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complementaryDNA (cDNA) strands. Ligase chain reaction, strand displacementamplification, self-sustained sequence replication, or nucleic acidsequence-based amplification also can be used to obtain isolated nucleicacids. See, for example, Lewis (1992) Genetic Engineering News 12:1;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; andWeiss (1991) Science 254:1292.

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Isolated nucleic acids (e.g., nucleic acids encoding a polypeptideprovided herein) also can be obtained by mutagenesis. For example, areference sequence (e.g., SEQ ID NO:2 or 4) can be mutated usingstandard techniques including oligonucleotide-directed mutagenesis andsite-directed mutagenesis through PCR. See, Short Protocols in MolecularBiology, Chapter 8, Green Publishing Associates and John Wiley & Sons,edited by Ausubel et al., 1992.

Vectors containing nucleic acids such as those described herein also areprovided. A “vector” is a replicon, such as a plasmid, phage, or cosmid,into which another DNA segment may be inserted so as to bring about thereplication of the inserted segment. An “expression vector” is a vectorthat includes one or more expression control sequences, and an“expression control sequence” is a DNA sequence that controls andregulates the transcription and/or translation of another DNA sequence.

In the expression vectors, a nucleic acid (e.g., a nucleic acid encodinga polypeptide provided herein) can be operably linked to one or moreexpression control sequences. As used herein, “operably linked” meansincorporated into a genetic construct so that expression controlsequences effectively control expression of a coding sequence ofinterest. Examples of expression control sequences include promoters,enhancers, and transcription terminating regions. A promoter is anexpression control sequence composed of a region of a DNA molecule,typically within 100 to 500 nucleotides upstream of the point at whichtranscription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it is necessary to position the translation initiation site ofthe translational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of expression control sequences in acell when RNA polymerase is able to transcribe the coding sequence intomRNA, which then can be translated into the protein encoded by thecoding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. In somecases, a viral vector can be virus particles such as type fiveadenovirus, helper-dependent adenovirus, adeno associated virus, measlesvirus, or lentivirus virus particles that are designed to express a wildtype BChE polypeptide or mutant BChE polypeptide provided herein.Numerous vectors and expression systems are commercially available fromsuch corporations as Novagen (Madison, Wis.), Clontech (Palo Alto,Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

This document also provides host cells containing a nucleic acid orvector provided herein. The term “host cell” is intended to includeprokaryotic and eukaryotic cells into which a recombinant nucleic acidor vector (e.g., an expression vector) can be introduced. As usedherein, “transformed” and “transfected” encompass the introduction of anucleic acid molecule (e.g., a vector) into a cell by one of a number oftechniques. Although not limited to a particular technique, a number ofthese techniques are well established within the art. Suitable methodsfor transforming and transfecting host cells can be found, for example,in Sambrook et al., Molecular Cloning: A Laboratory Manual (2^(nd)edition), Cold Spring Harbor Laboratory, New York (1989). For example,calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transfercan be used introduce nucleic acid into cells. In addition, naked DNAcan be delivered directly to cells in vivo as described elsewhere (U.S.Pat. Nos. 5,580,859 and 5,589,466).

Compositions and Methods for Administration

A wild type BChE polypeptide or mutant BChE polypeptide provided herein,or a nucleic acid encoding a wild type BChE polypeptide or mutant BChEpolypeptide provided herein, can be incorporated into a composition foradministration to a mammal (e.g., an obese or aggressive human who isseeking treatment). For example, a viral vector designed to express awild type BChE polypeptide or a mutant BChE polypeptide provided hereincan be administered to a mammal (e.g., a human) under conditions whereinthe body weight of the mammal, the rate of weight gain, and/or the levelof aggressiveness of the mammal is reduced in a therapeutic manner.Compositions containing a wild type BChE polypeptide or mutant BChEpolypeptide provided herein (or a nucleic acid encoding such apolypeptide) may be given once or more daily, weekly, monthly, or evenless often, or can be administered continuously for a period of time(e.g., hours, days, or weeks). In some cases, preparations designed tostabilize such polypeptides may maintain effective activity in a mammalfor several days. This document provides a viral vector or viral vectorsdesigned to express a natural or mutant BChE polypeptide providedherein, which can be administered once to a mammal in a way thatgenerates effective amounts of the polypeptide for months or years(e.g., two years or longer). In some cases, such treatment can beextended by later administration of an equivalent viral vector ofaltered serotype (e.g., type 8 adenoviral vector) to express the samepolypeptide for extended treatments.

The polypeptide or nucleic acid to be administered to a mammal can beadmixed, encapsulated, conjugated or otherwise associated with othermolecules, molecular structures, or mixtures of compounds such as, forexample, liposomes, receptor or cell targeted molecules, or oral,topical or other formulations for assisting in uptake, distributionand/or absorption. In some cases, a composition to be administered cancontain a polypeptide or nucleic acid in combination with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers include, for example, pharmaceutically acceptable solvents,suspending agents, or any other pharmacologically inert vehicles fordelivering polypeptides, nucleic acids, or viral vectors (e.g., viralparticles) to a subject. Pharmaceutically acceptable carriers can beliquid or solid, and can be selected with the planned manner ofadministration in mind so as to provide for the desired bulk,consistency, and other pertinent transport and chemical properties, whencombined with one or more therapeutic compounds and any other componentsof a given pharmaceutical composition. Typical pharmaceuticallyacceptable carriers include, without limitation: water; saline solution;binding agents (e.g., polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose or dextrose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

Acceptable solvents for delivery of viral vectors include, withoutlimitation, common physiological salt solutions such as 0.9% sodiumchloride, or isotonic aqueous solutions of sodium phosphate buffered toa pH of 7.4.

Pharmaceutical compositions containing a polypeptide, nucleic acid, orviral vector as described herein can be administered by a number ofmethods including by subcutaneous, intrathecal, intraventricular,intramuscular, intraperitoneal, or intravenous injection.

The invention will be further described in the following example, whichdoes not limit the scope of the invention described in the claims.

EXAMPLES Example 1 BChE Catalyzes Ghrelin Hydrolysis

A sample of purified human BChE was obtained from Dr. O. Lockridge andassessed by gel electrophoresis (FIG. 3A). The enzyme preparationresulted in a primary band for monomeric BChE and a secondary band fordimeric BChE (FIG. 3A). No contaminants were detected. Incubation of asample containing acyl ghrelin with increasing amounts of human BChEresulted in a decrease in the amount of acyl ghrelin and an increase inthe amount of the breakdown product, desacyl ghrelin (FIG. 3B). A linearhydrolysis rate was observed (FIG. 3C). The hydrolysis of acyl ghrelinby human BChE was blocked by a BChE selective inhibitor (iso-OMPA) atthe same concentration that blocks butyrylthiocholine (BTCh) hydrolysis(FIG. 3D). No inhibition of acyl ghrelin hydrolysis was observed usingprotease inhibitors (FIG. 3D). This is an example confirming that BChEhas a well-developed capacity to metabolize ghrelin.

Example 2 In Vitro Screening Mutant Butyrylcholinesterases

Mutations of BChE

Human and mouse butyrylcholinesterase cDNAs were subjected to a seriesof amino acid substitutions in the region of the active site. Briefly,the steps were as follows. First, wild type mouse BChE cDNA or humanBChE was cloned into a pAAV-CMV shuttle plasmid for a serotype 8adeno-associated virus (AAV) gene transfer vector. A Kozak consensussequence (GCCACC) was introduced before the translational start site.With this construct as template, site-directed mutagenesis using primerswith specific base-pair alterations generated the desired sequences. Thefollowing mutants were made:

-   -   1. A328W (vs. human)    -   2. A328W/Y332A (vs. human)    -   3. F227A/S287G/A328W/Y332M (vs. human)    -   4. S227A/S287G/A328W/Y332M (vs. mouse)    -   5. A199S/A328W/Y332G (vs. human)    -   6. A199S/F227A/S287G/A328W/Y332G (vs. human BChE)    -   7. A199S/S227A/S287G/A328W/Y332G (vs. mouse BChE)    -   8. A199S/S287G/A328W/Y332A (vs. human)    -   9. F227A/S287G/A328W/Y332G (vs. human)    -   10. A199S/S287G/A328W/Y332G (vs. human)    -   11. F227A/S287G/A328W/Y332A (vs. human)    -   12. A199S/F227A/S287G/A328W/E441D (vs. human)    -   13. A199S/S227A/S287G/A328W/E441D (vs. mouse)    -   14. A199S/F227A/A328W/Y332G (vs. human)    -   15. A199S/F227A/S287G/A328W/Y332G/E441D (vs human)    -   16. A199S/S227A/S287G/A328W/Y332G/E441D (vs mouse)    -   17. F329M (vs. both)    -   18. A199S/F227A/S287G/A328W/F329M/Y332G (vs. human BChE)    -   19. A199S/S227A/S287G/A328W/F329M/Y332G (vs. mouse BChE)        Enzyme Screening Method

The A199S/F227A/S287G/A328W/Y332G (mutant human BChE) and theA199S/S227A/S287G/A328W/Y332G (mutant mouse BChE) enzymes were tested invitro. For in vitro testing, HEK293 cells were transduced with AAVvector encoding the relevant enzyme cDNA, and enzyme was purified fromculture supernatants by procainamide Sepharose column chromatographyfollowed by ion-exchange chromatography. Purification led to a singlemajor band on SDS polyacrylamide gel. Active sites were titrated withDFP to determine final molar enzyme concentration as described elsewhere(Geng et al., PloS-One, 8(6)e67446 (2013)). Samples were then testedwith a commercial human ghrelin immunoassay kit to determine ghrelinhydrolyzing activity in vitro according to (a) a decrease inimmunoreactive acyl ghrelin and (b) an increase in immunoreactive deacylghrelin.

The A199S/F227A/S287G/A328W/Y332G mutant human BChE exhibited modestactivity, comparable to that of wild type human BChE, while theA199S/S227A/S287G/A328W/Y332G mutant mouse BChE exhibited enhancedactivity (e.g., at least 20-fold more activity), comparable to that ofwild type mouse BChE.

Example 3 Expression of a Mutant BChE In Vivo Reduces Acyl GhrelinLevels and Increases Desacyl Ghrelin Levels

Method for Viral Vector Delivery of Recombinant Butyrylcholinesterase toMice

Standard methods were used to introduce BChE cDNA into mice via hdAD andAAV viral gene transfer vectors. To produce and purify AAV8 viralparticles, the plasmids pAAV-CMV-BChE (wt or mCocH) or pAAV-VIP-mCocHwere co-transfected into HEK293T cells with helper vectors, pHelper andpAAV2/8, using FuGene HD Transfection Reagent (Roche). Three days later,AAV8 virus was purified from the cell lysates by ultracentrifugationagainst Optiprep Density Gradient Medium-Iodixanol (Sigma-Aldrich, StLouis Mo.). The concentration of viral particles was subsequentlydetermined by real-time quantitative PCR (QPCR), which also was used toestablish the tissue distribution of delivered vector.

Mutated BChE also was incorporated into a serotype-5 helper dependentadenoviral vector (hdAD) under regulation by a human ApoE hepaticcontrol region (Kim et al., Proc. Natl. Acad. Sci. USA, 98:13282-13287(2001)), with a bovine growth hormone polyadenylation sequence clonedinto a derivative of the p281acZ hdAD-backbone plasmid. Vector waspropagated using the AdNG163 helper virus as described elsewhere (Parkset al., Proc. Natl. Acad. Sci. USA, 93:13565-13570 (1996)). Particletiters were then determined by optical density at 260 nm. Helper viruscontamination, determined by plaque assay on HEK-293 cells, was 0.2% forboth loaded and empty vectors.

Fed and fasting BALB/C mice (n=4) were injected with adeno-associatedviral (AAV) vectors designed to express luciferase (control; AAV-Luc),wild-type mouse BChE (AAV-mBChE wt), or a mutant mouse BChE (AAV-mBChEmut). Uninjected mice (n=4) also were used as a control. The mutantmouse BChE had the sequence set forth in FIG. 1 with the following fiveamino acid substitutions: A199S/S227A/S287G/A328W/Y332G Injection ofAAV-mBChE mut resulted in a reduction in the plasma levels of acylghrelin and an increase in the plasma levels of desacyl ghrelin (FIG.4). No significant effects were observed for the mice receiving AAV-Lucor AAV-mBChE wt (FIG. 4).

In another experiment, mice injected with either AAV-Luc or AAV-mBChEmut were injected intravenously with exogenous acyl ghrelin peptide (1mg/mouse), and the levels of acyl ghrelin in plasma were measured 1, 3,and 10 minutes later. Mice injected with AAV-mBChE mut exhibited anincreased ability to eliminate acyl ghrelin from plasma (FIG. 5).

In another experiment, plasma obtained from mice injected with eitherAAV-Luc or AAV-mBChE mut followed by injection with either saline oriso-OMPA was assessed for the ability to hydrolyze BTCh in vitro.Treatment with iso-OMPA abolished the BTCh hydrolysis activity observedin samples from mice receiving AAV-mBChE mut in the absence of iso-OMPA(FIG. 6). In addition, the level of acyl ghrelin in plasma for AAV-mBChEmut-treated mice receiving iso-OMPA was equivalent to the level observedin AAV-Luc-treated control mice (FIG. 6). These results demonstrate thatthe reduced acyl ghrelin levels in mice receiving gene transfer ofmutant BChE are specifically due to BChE-driven catalysis of the activepeptide.

In another experiment, C57b1/6 mice were injected with AAV-Luc vector,AAV-mBChE wild type, or AAV-mBChE mutant vector at the dose of 1×10¹³viral particles per mouse at 6-week age. Plasma samples were collectedat the indicated time points and assayed for BTCh hydrolysis activity,which was much higher in the samples from mice given BChE vectors thanin mice given the luciferase (control) vector (FIG. 7). All values aremeans ±SD, each with triplicate samples (n=5 per group).

These results demonstrate that BChE is capable of inactivating ghrelinand that appropriate mutations in the BChE active site can cause largeincreases in peptide hydrolyzing activity. These results alsodemonstrate that a high activity mutant can be expressed indefinitely inmice after a single injection of viral vector (2 years or more) and thatmice given such vector can have a 90% reduction in levels of activeghrelin in blood plasma with no detectable adverse effect. Further,injected exogenous ghrelin disappears much faster in vector-treated micethan in control mice, and selective inhibition of BChE can preventaccelerated ghrelin destruction and can raise ghrelin levels, butprotease inhibitors have no such effect.

Example 4 Expression of a Wild Type or Mutant BChE In Vivo Reduces BodyWeight Gain

Controls were C57BL6 mice treated with AAV luciferase vector or noinjection. The experimental group received either AAV wild type mouseBChE vector or AAV mutant mouse BChE vector as indicated in Example 3.Two sets of experimental and control mice were tested for weight gain(n=5 to 8). One set received normal laboratory mouse diet. The other setreceived a high fat diet with 45% of calories from fat over theobservation periods of 12 to 16 weeks. Results are provided in FIG. 8.

Example 5 Expression of Wild Type or Mutant BChE In Vivo ReducesAggressive Behavior

Controls were untreated or saline-treated mice (male Balb/C mice). Theexperimental group received the A199S/S227A/S287G/A328W/Y332G mutantmouse BChE by hdAD viral gene transfer (dose=1.7×10¹² viral particlesi.v.), delivered at about 6 weeks of age. The numbers of fights persession on successive trials in the standard “resident-intruder” modelwere scored by treatment-blind observer. Plasma BChE activity andghrelin levels (pre-fight) also were recorded.

Mice treated with the hdAD vector expressing mutant mouse BChE exhibitedsignificantly reduced fighting compared with saline treated controlstested at 14-16 months of age (FIG. 9; p=0.003 by 2-way ANOVA). Micetreated with AAV vector encoding the same mutant mouse enzyme tested at8 months of age also exhibited reduced fighting (FIG. 10; p=0.005).Plasma from these animals again revealed an about 100× increase in BChElevels and a substantial (50%) reduction in active ghrelin vs. salinecontrols and luciferase vector controls.

To prove that reduced ghrelin caused the reduce aggression, mice weretreated with a mutant human BChE (“Mut 6 with C terminal truncation) AAVVIP mut6-C (10e13pt/mouse, n=10) that was poor at inactivating ghrelin.These mice were assessed using the aggression test at 15 to 16 month andexhibited no group difference with control (FIG. 11). Plasma from theseanimals exhibited a large increase in BChE activity vs.butyrylthiocholine (94-fold above control) and cocaine (still largerincrease), but no reduction of acyl ghrelin (only a non-significant 15%decrease). The enzyme assays revealed a nearly 100-fold increase in BChEand a 92% drop in active ghrelin (Table 2). These results demonstratethat BChE-catalyzed loss of active ghrelin is involved in theanti-aggression effect of BChE gene transfer.

TABLE 2 Levels of active ghrelin (acyl ghrelin) and BChE activity inplasma samples collected at indicated ages from mice treated at about 6weeks of age with the indicated viral gene-transfer expression vectorsencoding the indicated enzymes. Controls received saline injections orvector encoding irrelevant protein (luciferase). Fasting BChE levelacyl-ghrelin (U/mL) Vector Mean % Mean x-fold vs Age treatment n (pg/ml)control (U/ml) control ~15 month hd-AD 7  62 ± 8.9  7.7%  60 ± 6.7 82Mut 1 m-BChE ~15 month Saline 10  801 ± 310 —   0.7 ± 0.03 10 monthAAV-8 10  177 ± 8.0  85%  18 ± 3.5 94 Mut6- h-BChE 10 month saline 13210 ± 18 —  0.2 ± 0.06 8 month AAV-8 5  78 ± 10  52% 197 ± 23 123 m-BChE8 month Saline 7 339 ± 59 —  1.6 ± 0.04 3 month None 910 ± 65 154% 0.010% (BChE knockout) 1 month AAV-WT 5 308 ± 62  53%  558 ± 183 299 hBChE 3× 10{circumflex over ( )}12 1 month AAV-F329M 2  109 ± 17*  19% 480 ± 77257 Mut hBChE 3 x 10{circumflex over ( )}12 1 month AAV-F329M 8  14 ±8.5   8% 592 ± 80 540 Mut mBChE 10{circumflex over ( )}13 1 month saline5  581 ± 120   1.9 ± 0.27 1.9

In another experiment, three month old C57/BL6 BChE knockout mice weretested for aggression. The knockout mice exhibited no detectable BChEand exhibited moderate elevation of ghrelin levels in the fed state(normal condition) and significantly higher aggression than wild-typemice of the same strain (FIG. 12).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A polypeptide having the amino acid sequence setforth in SEQ ID NO:3 with a F329M substitution or with a combinationwith one or more of the following amino acid substitutions: A199S,F227A, S287G, A328W, or Y332G, wherein said polypeptide has an increasedability to hydrolyze acyl ghrelin as compared to a polypeptide havingthe amino acid sequence set forth in SEQ ID NO:3.
 2. The polypeptide ofclaim 1, wherein said polypeptide comprises said amino acid sequencecomprising said F329M substitution.
 3. The polypeptide of claim 1,wherein said polypeptide comprises said amino acid sequence comprisingsaid A199S, F227A, S287G, and A328W substitutions.
 4. The polypeptide ofclaim 1, wherein said polypeptide comprises said amino acid sequencecomprising said A199S, F227A, S287G, A328W, and Y332G substitutions. 5.The polypeptide of claim 1, wherein said polypeptide comprises saidamino acid sequence comprising said A199S, F227A, S287G, A328W, andF329M substitutions.
 6. The polypeptide of claim 1, wherein saidpolypeptide comprises said amino acid sequence comprising said A199S,F227A, S287G, A328W, F329M, and Y332G substitutions.